Compounds for protecting hydroxyls and methods for their use

ABSTRACT

A hydrocarbyldithiomethyl-modified compound of the Formula: 
     R 1 —O—CH 2 —S—S—R 2   
     or a salt thereof wherein R 1  is an organic molecule and R 2  is a hydrocarbyl is useful for protecting and/or blocking hydroxyl groups in organic molecules such as nucleotides. The hydrocarbyldithiomethyl-modified compounds can also be used for chemically synthesizing oligonucleotides and for sequencing nucleic acid compounds.

FIELD OF THE INVENTION

[0001] The invention relates to biological chemistry in general. Inparticular, the invention relates to protecting hydroxyls in organicmolecules.

BACKGROUND OF THE INVENTION

[0002] Temporary protection or blocking of chemically reactive functionsin biological compounds is an important tool in the field of biologicalchemistry. To this end, researchers have developed a number ofprotecting groups. The vast majority of the known protecting groups,however, are acid or base labile and while there are also protectinggroups that are labile under neutral conditions, most of theseprotecting groups are also somewhat acid and base labile. Greene, T W,“Protective Groups in Organic Synthesis”, publishers Wiley-Interscience(1981). Furthermore, many protecting groups suffer additional synthesis,side-reaction, and/or solubility problems. For example, only a fewprotecting groups applied as a part of a linking system between thesolid phase and the oligonucleotide can withstand all the rigors ofoligonucleotide synthesis and deprotection thereby facilitating thefinal purification of oligonucleotides free of truncated or depurinatedfragments. See “Solid Phase Synthesis,” Kwaitkowski et al., PCTInternational Publication WO 98/08857 (1996). Selective post-syntheticderivatization of oligonucleotides also requires selectively cleavableprotecting groups. See, e.g., Kahl & Greenberg, “Introducing StructuralDiversity in Oligonucleotides via Photolabile, ConvertibleC5-substituted Nucleotides,” J. Am. Chem. Soc., 121(4), 597-604 (1999).

[0003] Protecting groups also should be removable. Ideally, theprotecting group is removable under mild conditions, for example,without disturbing interactions between biomolecules. These types ofprotecting groups may be useful for deprotecting oligonucleotideswithout disturbing interactions between oligo/polynucleotide strands.For example, International Publication WO 96/23807 entitled “Novel ChainTerminators, The Use Thereof for Nucleic Acid Sequencing and Synthesisand a Method of their Preparation” discloses methods that usenucleotides that are reversibly blocked at the 3′ hydroxyl group. Thesereversibly blocked nucleotides can be used in sequencing methods where,unlike the well-known Sanger sequencing method that utilizes terminatingdideoxynucleotides, the temporarily 3′-OH-protected intermediates can beconverted into nucleotides having a free 3′-OH that may be furtherextended.

[0004] One such sequencing method that uses reversibly blockednucleotides is known as Sequencing by Synthesis (SBS). SBS determinesthe DNA sequence by incorporating nucleotides and detecting the sequenceone base at a time. To effectively sequence long stretches of a nucleicacid using SBS, it is advantageous to be able to perform multipleiterations of the single nucleotide incorporation. Accordingly,SBS-based methods require 3′-OH protecting groups that are removableunder conditions that do not distrupt the primer and target DNAinteractions. As such, there exists a need for nucleotide triphosphatesthat are reversibly blocked at the 3′ position and which are alsoeffective substrates for DNA polymerases.

SUMMARY OF THE INVENTION

[0005] In one aspect, the invention provides ahydrocarbyldithiomethyl-modified compound of the Formula:

R¹—O—CH₂—S—S—R²

[0006] or a salt thereof, wherein R¹ is an organic molecule and R² is ahydrocarbyl. Before undergoing hydrocarbyldithiomethyl-modification, R¹has at least one hydroxyl group, which after modification is in an etherlinkage. In one embodiment, R² further includes a labeling group. Thelabeling group can be any type of labeling group including fluorescentlabeling groups, which can be selected from the group consisting ofbodipy, dansyl, fluorescein, rhodamin, Texas red, Cy 2, Cy 4, and Cy 6.

[0007] In another embodiment, R¹—O represents modified or unmodifiedamino acids, peptides, proteins, carbohydrates, sterols,ribonucleosides, ribonucleotides, base- and/or sugar-modifiedribonucleosides, base- and/or sugar-modified ribonucleotides,deoxyribonucleosides, deoxyribonucleotides, base- and/or sugar-modifieddeoxyribonucleosides, and base- and/or sugar-modifieddeoxyribonucleotides. R¹ can have more than one hydroxyl group and morethan one of the hydroxyl groups can be modified with ahydrocarbyldithiomethyl moiety. For nucleotide embodiments, thehydrocarbyldithiomethyl modification can be at the 2′ and/or, 3′, and/or5′ hydroxyl positions of the R¹—O.

[0008] In another embodiment, R² includes a function that modifies theelectron density of the dithio function, thereby modifying the stabilityof the dithiol. Such a function may be provided by a chemical groupcontaining elements selected from the group consisting of oxygen,nitrogen, sulfur, and silicon.

[0009] In another aspect, the invention provides ahydrocarbyldithiomethyl-modified compound of the Formula:

[0010] or a salt thereof, wherein R¹ is H, a protecting group,phosphate, diphosphate, triphosphate, or residue of a nucleic acid, R²is a nucleobase, R³ is H, OH, or a protected form of OH; and R⁴, R⁵ andR⁶ are together or separately H, hydrocarbyl, or a residue of a solidsupport. Suitable hydrocarbyls for R⁴, R⁵ and R⁶ include methyl, ethyl,isopropylgy, and t-butyl. In one embodiment, R⁴, R⁵ and R⁶ together orseparately further include a labeling group and/or an electron donatingfunction or electron density modifying function. The electron densitymodifying function can be a heteroatom selected from the groupconsisting of oxygen, nitrogen, sulfur, and silicon.

[0011] In another aspect, the invention provides a compound of theFormula:

[0012] or a salt thereof, wherein R¹ is H, a protecting group, aphosphate, diphosphate, or a triphosphate, or a residue of a nucleicacid, R² is nucleobase, R³ is H or OH, or a protected form of OH, and R⁴is H or hydrocarbyl. In one embodiment, R⁴ is modified with a labelinggroup. In other embodiments, R⁴ includes a derivatizable function, or R⁴includes nitrogen, or R⁴ is covalently linked to a solid support.

[0013] In another aspect, the invention provides a method for modifyinga nucleoside including the steps of: a) contacting a nucleoside havingat least one hallogenomethyl-modified hydroxyl group with anthiosulfonate compound thereby forming a thiosulfonated nucleoside; andb) contacting the thiosulfonated nucleoside with a hydrocarbylthiolcompound thereby forming a hydrocarbyldithiomethyl-modified nucleoside.Useful thiosulfonate compounds include alkylthiosulfonate andarylthiosulfonate.

[0014] In one embodiment, the method includes the step of labeling thehydrocarbyldithiomethyl-modified nucleoside.

[0015] In another aspect, the invention provides a method for sequencinga nucleic acid including the steps of: a) contacting a target nucleicacid with a primer wherein at least a portion of the primer iscomplementary to a portion of the target nucleic acid; b) incorporatinga hydrocarbyldithiomethyl-modified nucleotide into the primer; and c)detecting incorporation of the hydrocarbyldithiomethyl-modifiednucleotide, wherein the hydrocarbyldithiomethyl-modified nucleotide iscomplementary to the target nucleic acid at thehydrocarbyldithiomethyl-modified nucleotide's site of incorporation. Inone embodiment, the incorporating step is catalyzed by a DNA polymerase.Useful sequencing methods that may use the method disclosed aboveinclude minisequencing and sequencing by synthesis whether performed inisolation or performed as a sequencing array.

[0016] In another aspect, the invention provides a method for sequencinga nucleic acid including the steps of: a) contacting a target nucleicacid with a primer wherein at least a portion of the primer iscomplementary to a portion of the target nucleic acid; b) incorporatinga first hydrocarbyldithiomethyl-modified nucleotide into the primer; c)detecting the incorporation of the firsthydrocarbyldithiomethyl-modified nucleotide; d) removing thehydrocarbyldithiomethyl group from the first incorporatedhydrocarbyldithiomethyl-modified nucleotide to form a first elongatedprimer having a free hydroxyl group; e) incorporating a secondhydrocarbyldithiomethyl-modified nucleotide into the first elongatedprimer; and f) detecting the second hydrocarbyldithiomethyl-modifiednucleotide, wherein the first hydrocarbyldithiomethyl-modifiednucleotide and the second hydrocarbyldithiomethyl-modified nucleotideare complementary to the target nucleic acid at each nucleotide's siteof incorporation. Following the sequencing method steps once willidentify the sequence of one nucleobase of the target nucleic acid.Repeating the steps can facilitate identifying the sequence of more thanone nucleobase of the target nucleic acid. The conditions of thesequencing method should be such that the primer anneals or hybridizesto the target nucleic acid in a sequence specific manner. In someembodiments the detecting steps are performed before removing thehydrocarbyldithiomethyl group whereas in other embodiments the detectingthe incorporation steps are performed after removing thehydrocarbyldithiomethyl group. In some embodiments, the method isoptimized for implementing the method in a sequencing array.

[0017] In another aspect, the invention provides a compound of theFormula:

[0018] wherein R¹ is a H, a protecting group, a phosphate, diphosphate,or a triphosphate, or a residue of a nucleic acid; R² is a nucleobase;R⁴, R⁵ and R⁶ are together or separately H or hydrocarbyl; and R⁷ is H,H-phosphonate or phosphoramidite.

[0019] In another aspect, the invention provides an oligonucleotidesynthesis support of the formula:

[0020] wherein R¹ is H, phosphate, diphosphate, triphosphate, or aprotecting group, R² is a nucleobase, R³ is H, OH, or a protected formof OH, and Z is a group effective for covalent attachment to a solidsupport, the solid support being effective for securing anoligonucleotide during oligonucleotide synthesis. In some embodiments, Zis selected from the group consisting of amino, amido, ester, and ether.

[0021] In another aspect, the invention provides a method forsynthesizing an oligonucleotide including the steps of: a) providing a5′ protected first nucleoside secured to a solid support through alinker; b) deprotecting the first nucleoside at its 5′ position; c)covalently bonding another 5′ protected nucleoside to the firstnucleotide at the 5′ position of the first nucleoside; d) deprotectingthe another nucleoside at its 5′ position; and e) repeating steps c) andd) for incorporating additional protected nucleosides. For this aspectof the invention, the linker secures the first nucleotide to the solidsupport via a hydrocarbyldithiomethyl bond. This synthesizing method canbe optimized for manufacturing oligonucleotide arrays.

[0022] In some embodiments, the oligonucleotide synthesis method iseffective for inverting the oligonucleotide thereby forming anoligonucleotide having a free 3′ hydroxyl and being secured to a solidsupport via another position.

[0023] In another aspect, the invention provides a method forsynthesizing an oligoribonucleotide including the steps of: a) providinga first protected ribonucleoside covalently linked to a solid support;b) covalently linking at least one hydrocarbyldithiomethyl-modifiedribonucleoside to the first ribonucleoside forming anoligoribonucleotide; c) partially de-protecting the oligoribonucleotideunder acidic or basic conditions; and d) contacting theoligoribonucleotide with a reducing agent under neutral conditionsthereby completely de-protecting the oligoribonucleotide, wherein thehydrocarbyldithiomethyl-modified ribonucleoside includes ahydrocarbyldithiomethyl group bound at the 2′ position of thehydrocarbyldithiomethyl-modified ribonucleoside. Such a method iseffective for preventing cleavage or migration of intemucleotidephosphate bonds during deprotection and is also effective for invertingthe oligoribonucleotide thereby forming a solid phase boundoligonucleotide having a free 3′ hydroxyl. In some embodiments, the pHis neutral and can be about 7 or range from about 5 to about 9. In otherembodiments, the first protected ribonucleoside is secured or covalentlylinked to the solid support via a hydrocarbyldithiomethyl bond.

[0024] In another aspect, the invention provides a method for sequencinga nucleic acid including the steps of: a) providing a primer arrayincluding a plurality of sequencing primers; b) contacting a targetnucleic acid with the primer array thereby forming target-primercomplexes between complementary portions of the sequencing primers andthe target nucleic acid; c) incorporating a firsthydrocarbyldithiomethyl-modified nucleotide into at least one sequencingprimer portion of the target-primer complexes, the firsthydrocarbyldithiomethyl-modified nucleotide being complementary to thetarget nucleic acid; and d) detecting the incorporation of the firsthydrocarbyldithiomethyl-modified nucleotide, wherein the firsthydrocarbyldithiomethyl-modified nucleotide is complementary to thetarget sequence at the first hydrocarbyldithiomethyl-modifiednucleotide's site of incorporation. In one embodiment, the methodfurther includes the steps of: e) removing the hydrocarbyldithiomethylgroup from the first incorporated hydrocarbyldithiomethyl-modifiednucleotide to form a first elongated target-primer complex having a free3′ hydroxyl group; f) incorporating a secondhydrocarbyldithiomethyl-modified nucleotide into the first elongatedtarget-primer complex; and g) detecting the secondhydrocarbyldithiomethyl-modified nucleotide, wherein the secondhydrocarbyldithiomethyl-modified nucleotide is complementary to thetarget sequence at the second hydrocarbyldithiomethyl-modifiednucleotide's site of incorporation. As with other methods describedherein, the detecting step can be performed before or after removing ahydrocarbyldithiomethyl moiety. This sequencing method is effective forproducing a plurality of nucleotide sequences wherein the nucleotidesequences correspond to overlapping nucleotide sequences of the targetnucleic acid.

[0025] In another aspect, the invention provides a method for sequencinga nucleic acid including the steps of: a) providing a target nucleicacid array including a plurality of target nucleic acids; b) contactinga sequencing primer with the target nucleic acids hereby formingtarget-primer complexes between complementary portions of the sequencingprimers and the target nucleic acids; c) incorporating a firstydrocarbyldithiomethyl-modified nucleotide into at least one sequencingprimer portion of the target-primer complexes, the firsthydrocarbyldithiomethyl-modified nucleotide being complementary to thetarget nucleic acid; and d) detecting the incorporation of the firsthydrocarbyldithiomethyl-modified nucleotide, wherein the firsthydrocarbyldithiomethyl-modified nucleotide is complementary to thetarget sequence at the first hydrocarbyldithiomethyl-modifiednucleotide's site of incorporation. As with other methods describedherein, the detecting step can be performed before or after removing ahydrocarbyldithiomethyl moiety. This sequencing method is effective forproducing a plurality of nucleotide sequences wherein the nucleotidesequences correspond to overlapping nucleotide sequences of the targetnucleic acid.

[0026] In another aspect, the invention provides a method forsynthesizing an oligonucleotide that includes the steps of: a) providinga 5′ protected first nucleoside covalently bonded to a solid supportthrough a hydrocarbyldithiomethyl containing linker; b) deprotecting thefirst nucleoside at its 5′ position; c) covalently bonding another 5′protected nucleoside to the first nucleotide at the 5′ position of thefirst nucleoside; d) deprotecting the another nucleoside at its 5′position; e) optionally repeating steps c) and d) for adding additionalprotected nucleosides thereby producing an oligonucleotide; f)optionally selectively cleaving a protecting group from theoligonucleotide thereby forming a partially deprotected oligonucleotide;g) selectively cleaving the hydrocarbyldithiomethyl containing linker;and h) isolating the partially deprotected oligonucleotide. In oneembodiment, the method further includes the step of modifying the 3′terminus with a reactive or detectable moiety. In another embodiment, atleast one of the 5′ protected nucleosides contains ahydrocarbyldithiomethyl moiety.

[0027] Advantages of the invention include introducing temporary orreversible mutations in proteins, facilitating continuous sequencingmethods, blocking reactive species during chemical syntheses, maskingchemical groups for manufacturing purposes, using hydroxyl groups tointroduce labeling groups into organic molecules, and other similaruses. It is to be understood that particular embodiments of theinvention described herein may be interchanged with other embodiments ofthe invention.

[0028] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used topractice the invention, suitable methods and materials are describedbelow. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

[0029] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

DETAILED DESCRIPTION

[0030] The present invention relates to hydrocarbyldithiomethyl-modifiedcompounds having modified and/or protected hydroxyl groups, methods formanufacturing such compounds, and methods for their use. The generalformula for the hydroxyl-modifying moiety contains a dithiol. Thedithiol is designed so that modified hydroxyls can be de-protected underneutral conditions using mild reducing agents.

[0031] A hydrocarbyldithiomethyl-modified compound is shown in Formula1:

R¹—O—CH₂—S—S—R²  (Formula 1)

[0032] wherein R¹ represents any organic molecule that had at least onefree hydroxyl group before undergoing the hydrocarbyldithiomethylmodification. In Formula 1, “O” is the oxygen atom of the hydroxylgroup, which is now protected in an ether linkage. For example, beforemodification, R¹ can be a modified or unmodified amino acid or analogthereof, oligonucleotide, peptide, protein, carbohydrate,deoxyribonucleoside, deoxyribonucleotide, ribonucleoside,ribonucleotide, base- and/or sugar-modified ribonucleoside, base- and/orsugar-modified deoxyribonucleoside, base- and/or sugar-modifiednucleotide, sterol or steroid, as long as the organic molecule selectedhas at least one hydroxyl group capable of being hydrocarbyldithiomethylmodified. As referred to herein, oligonucleotides refers to anynucleotide polymer including polymers of deoxyribonucleotides,ribonucleotides, nucleotide analogs and mixtures thereof.

[0033] When R¹—O is an organic molecule having more than one freehydroxyl group, any number of the free hydroxyls may be modified with ahydrocarbyldithiomethyl moiety. Alternatively, one or more of theadditional hydroxyl groups can be modified and/or protected with otherknown hydroxyl modifying compounds or left unmodified. Differentprotecting groups may be used to protect different hydroxyl groups.Useful protecting groups, other than the hydrocarbyldithiomethyl-basedgroups described herein, and methods for their use are known to those ofskill in the art and include fluorenylmethyloxycarbonyl (FMOC),4-(anisyl)diphenylmethyltrityl (MMTr), dimethoxytrityl (DMTr),monomethoxytrityl, trityl (Tr), benzoyl (Bz), isobutyryl (ib), pixyl(pi), ter-butyl-dimethylsilyl (TBMS), and1-(2-fluorophenyl)-4-methoxypiperidin 4-yl (FPMP). See, e.g., Greene, TW, “Protective Groups in Organic Synthesis”, publishersWiley-Interscience (1981); Beaucage & Iyer, “Advances in the Synthesisof Oligonucleotides by the Phosphoramidite Approach,” Tetrahedron,48(12):2223-2311 (1992); Beaucage & Iyer, “The Synthesis of SpecificRibonucleotides and Unrelated Phosphorylated Biomolecules by thePhosphoramidite Method,” Tetrahedron, 49(46):10441-10488 (1993); andScaringe et al., “Novel RNA Synthesis Method Using5′-O-silyl-2′-O-orthoester Protecting Groups,” J. Am. Chem. Soc.,120:11820-21 (1998). The choice of protective group can be dictated bythe type of organic molecule to be protected and the methods employed.Therefore, different organic molecules such as peptides,oligonucleotides, carbohydrates, and steroids may each use differentprotective groups. A hydroxyl with a known protecting group or ahydrocarbyldithiomethyl moiety attached to it can be referred to as aprotected form of the hydroxyl.

[0034] In Formula 1, R² represents a hydrocarbyl group. As used herein,hydrocarbyl groups include any organic radical having a carbon atomdirectly attached to the remainder of the molecule, e.g., saturated andunsaturated hydrocarbons, straight- and branched-chain aliphatichydrocarbons, cyclic hydrocarbons, aromatic hydrocarbons, heterocyclichydrocarbons, heteroaromatic hydrocarbons, and substituted hydrocarbonssuch as hydrocarbons containing heteroatoms and/or other functionalmodifying groups. The hydrocarbyl group may be covalently linked to asolid support (described below), labeling group or another organicmolecule.

[0035] Suitable hydrocarbons include alkyls (e.g., methyl, ethyl,propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl,dodecyl, tridecyl, and heptadecyl); alkoxy; alkenyl; C₃₋₈ alkenyloxy;alkynyl; alkynyloxy; C₃₋₂₀ cycloalkyl (e.g. cyclopropyl, cyclobutyl orcyclopentyl) in which the cycloalkyl may be substitutedn by one or morehydrocarbyls or heteroatoms; C₃₋₈ cycloalkoxy (e.g. cyclopentoxy); C₄₋₈cycloalkenyloxy (e.g. cyclopenten-3-yloxy); aryl (e.g. phenyl) oraralkyl (e.g. benzyl) in which the aryl may be substituted with one ormore C₁₋₄ alkyl, halogen, hydroxy, C₁₋₄ alkoxy, amino or nitro; aryloxy(e.g. phenoxy); arylalkoxy (e.g. benzyloxy) in which the aryl may besubstituted with one or more C₁₋₄ alkyl, halogen, hydroxy, C₁₋₄ alkoxy,amino or nitro; C₁₋₆ hydroxyalkyl (e.g. hydroxyethyl); and C₁₋₆alkoxyalkyl (e.g. methoxyethyl). In addition, all iso, sec and tertisomers of the aliphatic hydrocarbons are included such as isopropyl andt-butyl.

[0036] Substituted hydrocarbon groups are hydrocarbons containingnon-hydrocarbon substituents. Suitable substituents include oxygen,nitrogen, sulfur, phosphorous, halogens (e.g., bromine, chlorine,iodine, and fluorine), hydroxy, carbalkoxy (especially lower carbalkoxy)and alkoxy (especially lower alkoxy), the term, “lower” denoting groupscontaining 7 or less carbon atoms.

[0037] Other functional modifying groups capable of moderating thereactivity or lability of the disulfide bond or facilitate synthesizingcompounds of Formula 1 can be incorporated into R². Useful functionalmodifying groups are known and include heteroatoms such as oxygen,nitrogen, sulfur, phosphorous, and halogens. Functional modifying groupsalso include heterogroups such as amino, nitro, and cyano. These groupsmay function as an electron withdrawing or donating groups. Skilledartisans know whether electron withdrawing or donating groups would beappropriate.

[0038] R² may further include a labeling group. Useful labeling groupsare known to those of ordinary skill in the art and includeradioactively labeled groups, luminescent groups, electroluminescentgroups, fluorescent groups, and groups that absorb visible or infraredlight. Examples of useful fluorescent labels include bodipy, dansyl,fluorescein, rhodamin, Texas red, Cy 2, Cy 4, and Cy 6. Additionaluseful labels can be found in the “Handbook of Fluorescent probes andResearch Chemicals,” by Richard P. Haugland and “Nonisotopic DNA ProbeTechniques,” Ed. Larry J. Kricka (Academic Press, Inc. 1992).Hydrocarbyldithiomethyl-modified compounds can be created from availablecompounds using the following illustrative method. A iree hydroxyl on ahydroxyl-containing molecule is modified to form a methylthiomethylether. The methylthiomethyl ether can be formed by reacting the hydroxylwith a mixture of acetic anhydride, acetic acid and dimethyl sulfoxide(DMSO). See Hovinen et al., “Synthesis of3′-O-(ω-Aminoalkoxymethyl)thymidine 5′-Triphosphates, Terminators of DNAsynthesis that Enable 3′-Labeling,” J. Chem. Soc. Perkin Trans. I, pp.211-217 (1994). It is to be understood that, if the molecule to bemodified contains more than one hydroxyl group, it may be necessary tofirst protect or block one or more hydroxyl groups that are not to behydrocarbyldithiomethyl-modified.

[0039] The methylthiomethyl ether-derivatized compound is then convertedto a more reactive species such as a halogenatedmethyl ether. Usefulhalogens include bromine, chlorine, and iodine. The halogenation stepcan be carried out using any method including treating themethylthiomethyl ether with N-bromosuccinimide (NBS), or Br₂ in drychlorethane, or SOCl₂, or N-iodosuccinimide (NIS).

[0040] The halogenated methyl ether compound is then converted to ahydrocarbylthiolsulfonate reagent by treating it with an alkylhydrocarbylthiolsulfonate. See Bruice & Kenyon, “Novel AlkylAlkanethiolsulfonate Sulfhydryl reagents, Modification of Derivatives ofL-Cysteine,” J. Protein Chem., 1(1):47-58 (1982) and Plettner et al., “ACombinatorial Approach to Chemical Modification of Subtilisin Bacilluslentus,” Bioorganic & Medicinal Chem. Lett. 8, pp. 2291-96 (1998).

[0041] Contacting the hydrocarbylthiolsulfonate reagent with anyunsubstituted or substituted thiol can cause displacement of thesulfonyl moiety thereby creating a hydrocarbyldithiomethyl-modifiedcompound. Useful thiols include branched- and straight-chain aliphaticthiols, aromatic thiols, heteroaromatic thiols, substituted aliphaticthiols, functionally modified thiols, and fluorophore labeled thiols.Functionally modified thiols include thiols substituted at a carbon atomwith an atom or group capable of altering the reactivity of a dithiomoiety, capable of facilitating subsequent labeling, or capable offacilitating immobilization of the modified compound. Useful examples ofmodifying groups include amino, amido, hydroxyl, silyl, cyano,carboxylic esters, or other carboxylic substitutions.

[0042] It may be particularly useful to use the compounds of Formula 1when R¹ contains a nucleobase. As used herein, nucleobase includes anynatural nucleobase, synthetic nucleobase, and/or analog thereof. Naturalnucleobases include adenine, guanine, cytosine, thymine, uracil,xanthine, hypoxanthine, and 2-aminopurine. Synthetic nucleobases aretypically chemically synthesized and are analogues of the naturalnucleobases. Synthetic nucleobases are capable of interacting orhydrogen bonding with other nucleobases. Nucleobase containing compoundscan include both nucleosides and nucleotides. Nucleosides andnucleotides can be modified at the 5′, 3′ and/or 2′ hydroxyl positions.Known methods for protecting the 5′, 3′ and/or 2′ positions may be usedin conjunction with the methods described herein to modify individualhydroxyl positions.

[0043] For example, the compound shown in Formula 2

[0044] or a salt thereof can be synthesized using the methods describedherein. In Formula 2, R¹ is H, a protecting group, phosphate,diphosphate, triphosphate, or a residue of a nucleic acid; R² is anucleobase; R³ is H, OH, or a protected form of OH; R⁴, R⁵ and R⁶ aretogether or separately H, hydrocarbyl, or a residue of a solid support.For example, R⁴, R⁵ and R⁶ include together or separately H, methyl,ethyl, isopropyl, t-butyl, phenyl, or benzyl. It may be useful toinclude a substituted hydrocarbon having an electron density modifyinggroup containing a heteroatom or other functional modifying group atpositions R⁴, R⁵ or R⁶. For example, R⁴, R⁵ or R⁶ could bemethyleneamine, ethyleneamine, or contain an amino group. As an optionalaspect, R⁴, R⁵ or R⁶ can be modified with a labeling group.

[0045] Another illustrative example of usefulhydrocarbyldithiomethyl-modified compounds include the compounds ofFormula 3

[0046] or a salt thereof, wherein R¹ is H, a protecting group, aphosphate group, diphosphate group, or a triphosphate group; R² isnucleobase; R³ is H or OH, or a protected form of OH; and R⁴ is H , aheteroatom, a heterogroup, hydrocarbyl or a label-modified hydrocarbyl.R⁴ may be used to link the compound of Formula 3 to a solid support.

[0047] A nucleobase containing hydrocarbyldithiomethyl-modified compoundcan be chemically synthesized using the methods described herein. Forexample:

[0048] Compound A (wherein R¹ is a suitable protecting group, R² is anucleobase, and R³ is either a protected hydroxyl or H) is treated witha mixture of DMSO, acetic acid, and acetic anhydride to form amethylthiomethyl ether (compound B)

[0049] The methylthiomethyl ether (compound B) is converted to a morereactive halogenated species (compound C) wherein X is Br, Cl, or I.

[0050] Compound C is treated with an alkyl- or arylthiosulfonate such asmethylphenylthiosulfonate (MePhSO₂SH) to prepare compound D.

[0051] Compound D is treated with a hydrocarbylthiol compound such as2-thio-aminoethane to form a hydrocarbyldithiomethyl-modified nucleobase(compound E). In some instances, compound E may be the final product.

[0052] When the thiol used to form compound E contains a modifiablesubstituent, compound E can be further modified or labeled as shownbelow. Compound E can be treated under known conditions with anisothiocyanate form of a suitable fluorophore (such asfluoresceinisothiocyanate) to form compound F. Compound F can be furthermodified. For example, R¹ can be replaced with a mono, di, ortriphosphate group. Forming a triphosphate can facilitate using thenucleobase containing compounds in enzymatic and template-dependent DNAor RNA synthesis reactions.

[0053] Hydrocarbyldithiomethyl-modified nucleotide triphosphatesprotected at the 3′ position (as described above) are useful for anysequencing method. 3′-hydrocarbyldithiomethyl-modified nucleotidetriphosphates can terminate extension of the primer sequence when usedin a DNA polymerase-mediated sequencing method. Unlike most conventionaldideoxy methods where incorporation of the dideoxynucleotide ispermanent, however, termination using a hydrocarbyldithiomethyl-modifiednucleotide is reversible. Thus, one of the benefits associated withusing a hydrocarbyldithiomethyl-modified nucleotides for sequencing isthat the sequencing reaction can be stopped and started by utilizing thelabile nature of the protecting group. That is, thehydrocarbyldithiomethyl-moiety can be removed by reducing the disulfidebond of the protecting group. Reducing the disulfide creates an unstableintermediate that spontaneously decomposes to produce a free 3′hydroxyl, which can be used for attaching another nucleotide. Thedisulfide of the hydrocarbyldithiomethyl-moiety can be reduced using anyreducing agent. Suitable reducing agents include dithiothreitol (DTT),mercaptoethanol, dithionite, reduced glutathione, reduced glutaredoxin,reduced thioredoxin, and any other peptide or organic based reducingagent, or other reagents known to those of ordinary skill in the art.Reduction can be achieved under neutral conditions. It is to beunderstood that the reduction step leading to the spontaneousdecomposition of the intermediate is applicable to allhydrocarbyldithiomethyl-modified compounds. Accordingly, it may benecessary to adjust the conditions of conventional sequencing reactionsusing DNA polymerase enzymes that utilize reduced thioredoxin so thatfree thiols are not present when the hydrocarbyldithiomethyl-modifiednucleotides are added.

[0054] The compounds of Formulas 1-3 are useful as reagents in almostany method for sequencing a nucleic acid molecule. General methods forsequencing nucleic acids are known and include dideoxy sequencingmethods (Sanger et al., Proc. Natl. Acad. Sci., 74:5463-5467 (1977),chemical degradation methods (Maxam & Gilbert, Proc. Natl. Acad. Sci.,74:560-64 (1977), minisequencing methods (Syvänen et al., Genomics,8:684-92 (1990), and sequencing by synthesis (i.e., multiple iterationsof the minisequencing method). It is common practice in these methods toblock the 3′ hydroxyl of some of the nucleotides. Further, thesequencing by synthesis method requires the availability of nucleotideshaving a reversibly blocked 3′ hydroxyl.

[0055] For example, a sequencing method can proceed by contacting atarget nucleic acid with a primer. The target nucleic acid can be anynucleic acid molecule. The primer would also be a nucleic acid molecule.Typically, the primer is shorter than the nucleic acid to be sequenced.Methods for preparing nucleic acids for sequencing and for manufacturingand preparing primer sequences to be used in a sequencing reaction areknown. It is advantageous to design the primer so that at least aportion of the primer is complementary to a portion of the targetnucleic acid. It is also advantageous to design the primer so that thewhole primer is complementary to a portion of the target nucleic acid.

[0056] During a sequencing reaction, the primer and target nucleic acidsequences are combined so that the primer anneals or hybridizes to thetarget nucleic acid in a sequence specific manner. A DNA polymeraseenzyme is then used to incorporate additional nucleotides into theprimer in a sequence specific or template-dependent manner such that thenucleotide incorporated into the primer is complementary to the targetnucleic acid. For example, a 3′-hydrocarbyldithiomethyl-modifiednucleotide or a mixture of nucleotides is added to the sequencingreaction at a sufficient concentration so that the DNA polymeraseincorporates into the primer a single hydrocarbyldithiomethyl-modifiednucleotide that is complementary to the target sequence. Theincorporation of the hydrocarbyldithiomethyl-modified nucleotide can bedetected by any known method that is appropriate for the type of labelused.

[0057] A second or subsequent round of incorporation for thehydrocarbyldithiomethyl-modified nucleotide can occur after incubatingthe primer target sequence complex with a suitable reducing agent.Further, each round of incorporation can be completed without disruptingthe hybridization between primer and target sequence. After reduction ofthe disulfide, the 3′-OH becomes unblocked and ready to accept anotherround of nucleotide incorporation. The incorporation and reduction stepscan be repeated as needed to complete the sequencing of the targetsequence. In this way, it may be advantageous to differentially labelthe individual nucleotides so that incorporation of differentnucleotides can be detected. Such a method can be used in singlesequencing reactions, automated sequencing reactions, and array basedsequencing reactions.

[0058] Hydrocarbyldithiomethyl-modified deoxyribonucleotides andribonucleotides can also be used for synthesizing oligonucleotides.Chemical synthesis of oligoribonucleotides has an added complexitycompared to oligodeoxyribonucleotides due to the presence of the 2′-OHin ribonucleosides. The 2′-OH must be protected during synthesis.Further, the blocking group must be removable during final deblocking.Conditions used for deblocking conventional protecting groups canpromote cleavage and/or migration of intemucleotide linkages, i.e., the5′-3′ linkage of the oligonucleotide may migrate to form a 5′-2′linkage. This cleavage is both acid and base catalyzed, while migrationis acid catalyzed. As such, blocking the 2′-OH with ahydrocarbyldithiomethyl moiety is advantageous because the bond is: 1)stable under conventional/standard acidic and basic conditions whileother blocked regions of the oligoribonucleotide are deprotected, and 2)the hydrocarbyldithiomethyl moiety can be removed under neutralconditions using a simple reducing agent.

[0059] A method for synthesizing an oligoribonucleotide usingribonucleosides modified at the 2′-OH position with ahydrocarbyldithiomethyl moiety can proceed as follows. A firstnucleoside is linked to a solid support using known methods. See, e.g.,Pon, R T, “Chapter 19 Solid-phase Supports for OligonucleotideSynthesis,” Methods in Molecular Biology Vol. 20 Protocols forOligonucleotides and Analogs, 465-497, Ed. S. Agrawal, Humana PressInc., Towata, N.J. (1993). The 2′-OH modifying moiety can be ahydrocarbyldithiomethyl moiety or any other known protecting group(e.g., ester). Alternatively, the first ribonucleoside monomer is linkedto a solid support using known methods but also having a linker as shownin Formula 6 (described below). It is to be understood that the 5′-OHand the 3′-OH are also protected as needed using known methods or themethods described herein. After the initial nucleoside is tethered tothe solid support, additional hydrocarbyldithiomethyl-modifiedribonucleoside monomers are added to the growing oligoribonucleotideusing any of the existing strategies for intemucleotide bond formation.The completed oligoribonucleotide is then deblocked at all positionsexcept the 2′-O— position by using ammonia. The partially deblockedoligoribonucleotide is then contacted with a reducing agent underneutral conditions to achieve the final deprotection. Neutral conditionsare conditions that do not promote migration of internucleotidelinkages. Conditions having a pH value ranging from about 5 to about 9and any particular value therebetween, e.g., 7, are considered neutral.

[0060] A suitable ribonucleoside for use in a chemical oligonucleotidesynthesis reaction utilizing a hydrocarbyldithiomethyl-modifiedribonucleoside monomer is shown at Formula 4

[0061] wherein R¹ is a H or a protecting group, R² is a nucleobase, R⁴,R⁵ and R⁶ are together or separately H or hydrocarbyl. R⁷ may be H,H-phosphonate or phosphoramidite.

[0062] An example of Formula 4 is shown at Formula 5

[0063] wherein R¹ ² is a nucleobase, R⁴, R⁵ and R⁶ are together orseparately H or hydrocarbyl. Alternatively, other known blocking groupsmay be used to block the nucleoside at the 5′-OH and 3′-OH positionsaccording the needs of the skilled artisan.

[0064] Suitable ribonucleosides (as described above) can be preparedusing known methods and/or the methods described herein for adding ahydrocarbyldithiomethyl moiety to a nucleoside.

[0065] Chemical synthesis methods using the oligodeoxyribonucleotidesand oligoribonucleotides described herein can include methods forinverting the orientation of the oligonucleotides on the solid support.International Application WO 98/51698 entitled “Synthesis ofOligonucleotides” discloses methods for preparing immobilizedoligonucleotides and for their subsequent inversion to produceoligonucleotides having a free 3′-OH. These methods together with thecompounds and methods described herein can be used together to produceoligonucleotide arrays. The arrays are useful for binding and sequencingreactions, especially automated sequencing reactions.

[0066] Chemical synthesis methods using the oligodeoxyribonucleotidesand oligoribonucleotides described herein can include methods forpreparing oligonucleotides having different hydrophobic characteristics.Oligonucleotides can be designed to be more or less hydrophobic by usingselectively cleavable protecting groups. To alter the hydrophobiccharacter of the nucleotide, a subset of protecting groups is removedafter synthesizing the oligonucleotide. By altering the ratio ofprotecting groups attached the finished oligonucleotide to the number ofprotecting groups removed, the hydrophobicity of the finishedoligonucleotide can be controlled. These types of oligonucleotides canbe useful as pro-oligonucleotides for antisense drug treatment methodsfor a variety of disease states. The article Tosquellas et al., “ThePro-Oligonucleotide Approach: Solid Phase Synthesis And PreliminaryEvaluation Of Model Pro-Dodecathymidylates,” Nucleic Acids Res. 26:9,2069-74 (1998) provides an example of such pro-oligonucleotides.

[0067] Oligonucleotides having altered hydrophobicities can besynthesized by following the methods described herein. For example, afirst protected nucleoside is covalently attached to a solid support.Additional protected nucleosides are added according to methodsdescribed herein to assemble an oligonucleotide. The additionalnucleosides may each have different protecting groups. A subset of theprotecting groups can be removed. After reducing the dithiobond, theoligonucleotide is removed from the solid phase, washed out andcollected. This method allows for isolation of an almost completelyprotected oligonucleotide. The 3′-terminus of the oligonucleotide can bemodified with a reactive or detectable moiety. The oligonucleotidefragments activated at the 3′-position can be used for constructinglarger oligonucleotides or synthetic genes. They may also be used in amethod for combinatorial synthesis of gene variants lacking any unwantedstop codons.

[0068] Hydrocarbyldithiomethyl-modified compounds may also be used tolink molecules to a solid support. In particular, it may be advantageousto use hydrocarbyldithiomethyl-modified compounds for chemical synthesisof organic molecules such as oligonucleotides, peptides, andcarbohydrates. Several methods for coupling organic molecules to solidsupports are known. See, e.g., Pon, R T, “Chapter 19 Solid-phaseSupports for Oligonucleotide Synthesis,” Methods in Molecular BiologyVol. 20 Protocols for Oligonucleotides and Analogs, 465-497, Ed. S.Agrawal, Humana Press Inc., Towata, N.J. (1993). Only a few methods,however, provide linkages that are inert under acidic and basicconditions, and yield a free hydroxyl group after cleaving the linkage.For example, photochemically labile o-nitrobenzyl ether linkages,siloxyl linkages, and disiloxyl type linkages that are cleavable usingfluoride anions, are both inert under acidic and basic conditions andyield a free hydroxyl group after cleaving the linkage. Use of the abovelinkages is sometimes impractical or associated with unwanted sidereactions. The hydrocarbyldithiomethyl-modified linkages describedherein are cleavable under neutral conditions.

[0069] Accordingly, an oligonucleotide synthesis support can include themolecule shown in Formula 6

[0070] wherein R¹ is H, phosphate, diphosphate, triphosphate, or a5′-protecting group, R² is a nucleobase, R³ is H, OH, or a protectedform of OH, and Z is a group effective for covalent attachment to asolid support. Examples of Z include amido, ether and any other linkingfunction groups known to those of ordinary skill in the art. Such alinker capable of being coupled to a solid support can be effective forsecuring an oligonucleotide during oligonucleotide synthesis.

[0071] An example of a linker described in Formula 6 is shown in Formula7

[0072] wherein R² is a nucleobase, R³ is H, OH, or a protected form ofOH.

[0073] Chemical synthesis of an oligonucleotide can be done by attachinga first ucleoside monomer to a solid support. Any known solid supportcan be used including on-porous and porous solid supports and organicand inorganic solid supports. Useful solid supports includepolystyrenes, cross-linked polystyrenes, polypropylene, polyethylene,teflon, polysaccharides, cross-linked polysaccharides, silica, andvarious glasses. In some instances, certain solid supports are not fullycompatible with aspects of oligonucleotide synthesis chemistry. Forexample, strong alkaline conditions at elevated temperatures used fordeprotection of synthetic oligonucleotides or fluoride anions such asthose provided by tetrabutylammonium fluoride cannot be applied tosilica or glass supports. Conventional linkers and methods for attachingmonomers or oligonucleotides to a solid support are known. See Beaucage& Iyer, Tetrahedron, 48(12):2223-2311 (1992).

[0074] The invention will be fiurther described in the followingexamples, which do not limit the invention as set forth in the claims.

EXAMPLE 1 Synthesizing 5′-O-FMOC-Thymidine

[0075] Thymidine (10 mmol) was dried by coevaporation with dry pyridine(2×30 ml), re-dissolved in dry pyridine (50 ml) and cooled using anacetone/carbon dioxide bath to a temperature of −20° C. The thymidinesolution was magnetically stirred and a dichloromethane solution ofFMOC-Cl (12 mmol, 1.2 eq. in 20 ml DCM) was added over a period of 60minutes. The reaction mixture was warmed to room temperature and stirredfor additional 60 minutes. The reaction mixture was partitioned betweensaturated sodium hydrogen carbonate (250 ml) and dichloromethane (3×100ml). The organic phase was saved, combined, evaporated and dried bycoevaporation with toluene (2×50 ml) forming an oily residue. A pureproduct was crystallized from the oily residue using dichloromethane (30ml) and benzene (50 ml) as solvent. Yield 76%—white crystals.

EXAMPLE 2 Synthesizing 5′-O-FMOC-3′-O-methylthiomethyl-thymidine

[0076] The produce of Example 1 (5′-O-FMOC-Thymidine (7.0 mmol)) wasdissolved in 50 ml of an acetic acid:acetic anhydride:DMSO solution(11:35:54, v/v) at 20° C. according to (Zavgorodny et al. (1991)Tetrahedron Lett. 32:7593-7596). The solution was stirred at 20° C. for4 days resulting in a complete conversion of the starting material tomethylthiomethyl ether derivative as monitored by thin layerchromatography (TLC). The solvent was evaporated using a rotaryevaporator at 50° C. under high vacuum (oil pump). The residue wasdissolved in ethanol (30 ml) and poured into vigorously stirred water(500 ml). A solid material precipitated and was filtered off. Theprecipitate was then dissolved in dichloromethane, coevaporated withtoluene (2×50 ml), and flash chromatographed usingdichloromethane:chloroform (1:1 v:v) as the solvent to give the finalproduct as an oil. Yield 72%.

EXAMPLE 3 Synthesizing5′-O-FMOC-3′-O-(4-methylphenyltbiosulfonatemethyl)-thymidine

[0077] The product of Example 2(5′O-FMOC-3′-O-methylthiomethyl-thymidine (4.0 mmol)) was dissolved in asolution of dichloromethane (20 ml) and bromine (Br₂) (226 μl) was addedat 20° C. After a 10 minute incubation, a potassium salt ofp-toluenethiosulfonic acid (10.0 mmol) dissolved in dry DMF (10 ml) andlutidine (1.5 ml) was added. The reaction mixture was stirred for anadditional 120 minutes, quenched by addition of saturated NaHCO₃ andextracted with dichloromethane (3×50 ml). The resulting organic phasewas evaporated, coevaporated with toluene, and flash chromatographedusing chloroform as the final solvent. The final product was isolated asan oil. Yield 58%.

EXAMPLE 4 Synthesizing 3′-O-hydrocarbyldithiomethyl)thymidineDerivatives

[0078] The product of Example 3 (1 mmol) is dissolved in pyridine (5.0ml) and an appropriate thiol, such as reduced cystamine (“R”SH, i.e.,hydrocarbylthiol) (1.1 mmol) dissolved in pyridine (2.0 ml), is added.

[0079] The mixture is stirred for 60 min at 20° C., then extracted usingconventional bicarbonate extraction methods and purified by flashchromatography. In some instances it may be advantageous to continue thesynthetic process by addition of dry triethylamine (4.0 mmol) in orderto remove the 5′-O-FMOC protecting group. After 45 minutes the solventis evaporated and the 5′-OH derivative is isolated by chromatographyafter the standard work-up using aqueous NaHCO₂ and dichloromethane andevaporating the organic extracts.

EXAMPLE 5 Synthesizing 3′-O-(2-N-dansylethyldithiomethyl)-thymidine

[0080] The general procedure of Example 4 is followed usingN-dansylethanethiol as the thiol. N-dansylethanethiol is prepared byreacting cystamine dihydrochloride with dansyl chloride followed byreducing the disulfide with sodium borohydride. N-dansylethanethiol isisolated using rapid silica gel purification.

[0081] After forming the desired 2-N-dansylethyldithiomethyl linkage,the 5′ FMOC group is removed using known methods.

EXAMPLE 6 Synthesizing3′-O-(2-N-dansylethyldithiomethyl)-thymidine-5′-triphosphatetetralithium Salt

[0082] The product of Example 5(3′-O-(2-N-dansylethyldithiomethyl)-thymidine (0.1 mmol)) is dried bycoevaporation with dry pyridine (2×5 ml) and dissolved in dryacetonitrile (2.0 ml). Phosphorotristriazolide (0.1 M) in dryacetonitrile is prepared from phosphorus oxychloride and triazole asdescribed in Kraszewski & Stawinski, Tetrahedron Lett., 21:2935-2936(1980). Phosphorotristriazolide (1.5 ml, 1.5 eq.) is added to the3′-O-(2-N-dansylethyldithiomethyl)-thymidine at room temperature or 20°C. The mixture is stirred for 5 minutes at which point n-butylammoniumpyrophosphate in dry DMF (0.2 M, 1.5 ml, 2.0 eq.) is added. The mixtureis stirred overnight at 20° C. Water (2 ml) is then added and hydrolysisof the phosphates occurs (180 min). The nucleotide triphosphate isapplied to an anion exchange column Mono Q (TM) (Pharmacia Biotech.Sweden) equilibrated with triethylammonium bicarbonate (TEAB) (0.01 M)and eluted from the Mono Q (TM) column using a linear gradient of TEAB(0.85M):acetonitrile (33 %, v/v). The isolated3′-O-(2-N-dansylethyldithiomethyl)-thymidine-5′-triphosphatetetralithium salt fraction is evaporated, coevaporated with water andpassed through a Dowex 50W×8 (BDH) in a lithium form to accomplishexchange of the triethylamnionium to the lithium ions. At this point thehydrocarbyldithiomethyl-modified nucleotide is ready to be used.

[0083] Other aspects, advantages, and modifications are within the scopeof the following claims.

What is claimed is:
 1. A hydrocarbyldithiomethyl-modified compoundcomprising the Formula: R¹—O—CH₂—S—S—R² or a salt thereof, wherein R¹ isan organic molecule; and R² is a hydrocarbyl.
 2. The compound of claim 1wherein said R² comprises a fluorescent labeling group.
 3. The compoundof claim 2 wherein said fluorescent labeling group is selected from thegroup consisting of bodipy, dansyl, fluorescein, rhodamin, Texas red, Cy2, Cy 4, and Cy
 6. 4. The compound of claim 1 wherein said R¹ furthercomprises at least one hydroxyl group that is nothydrocarbyldithiomethyl-modified.
 5. The compound of claim 1 whereinsaid R¹ is selected from the group consisting of modified or unmodifiedamino acids, peptides, proteins, carbohydrates, sterols, or steroids. 6.The compound of claim 5 wherein said R² comprises a labeling group. 7.The compound of claim 5 wherein said R¹ further comprises at least onehydroxyl group that is not hydrocarbyldithiomethyl-modified.
 8. Thecompound of claim 1 wherein said R¹ is selected from the groupconsisting of ribonucleosides, ribonucleotides, base- and/orsugar-modified ribonucleosides, base- and/or sugar-modifiedribonucleotides, deoxyribonucleosides, deoxyribonucleotides, base-and/or sugar-modified deoxyribonucleosides, and base- and/orsugar-modified deoxyribonucleotides.
 9. The compound of claim 8 whereinsaid R² comprises a labeling group.
 10. The compound of claim 8 whereinsaid R¹ further comprises at least one hydroxyl group that is nothydrocarbyldithiomethyl-modified.
 11. The compound of claim 8 whereinsaid hydrocarbyldithiomethyl modification is at a 3′ hydroxyl positionof said R¹.
 12. The compound of claim 8 wherein saidhydrocarbyldithiomethyl modification is at a 5′ hydroxyl position ofsaid R¹.
 13. The compound of claim 8 wherein said R¹ is selected fromthe group consisting of ribonucleosides, ribonucleotides, base- and/orsugar-modified ribonucleosides, and base- and/or sugar-modifiedribonucleotides, and wherein said hydrocarbyldithiomethyl modificationis at a 2′ hydroxyl position of said R¹.
 14. The compound of claim 1wherein said R² comprises an electron donating or withdrawing function.15. The compound of claim 14 wherein said electron donating orwithdrawing function contains a heteroatom selected from the groupconsisting of oxygen, nitrogen, sulfur, and silicon.
 16. Ahydrocarbyldithiomethyl-modified compound comprising the formula:

or a salt thereof, wherein R¹ is H, a protecting group, phosphate,diphosphate, triphosphate, or residue of a nucleic acid; R² is anucleobase; R³ is H, OH, or a protected form of OH; and R⁴, R⁵ and R⁶are together or separately H, hydrocarbyl, or a residue of a solidsupport.
 17. The compound of claim 16 wherein R⁴, R⁵ and R⁶ together orseparately further comprise a labeling group.
 18. The compound of claim16 wherein R⁴, R⁵ and R⁶ comprise together or separately an electrondonating or withdrawing function.
 19. The compound of claim 18 whereinsaid electron donating or withdrawing function contains a heteroatomselected from the group consisting of oxygen, nitrogen, sulfur, andsilicon.
 20. The compound of claim 16 wherein R⁴, R⁵ and R⁶ are togetheror separately H, methyl, ethyl, isopropyl, t-butyl, phenyl, or benzyland wherein either R⁴, R⁵ or R⁶ is modified with a labeling group.
 21. Acompound comprising the Formula:

or a salt thereof, wherein R¹ is H, a protecting group, a phosphate,diphosphate, or a triphosphate, or a residue of a nucleic acid; R²is anucleobase; R³ is H or OH, or a protected form of OH; and R⁴ is H orhydrocarbyl.
 22. The compound of claim 21 wherein R⁴ is modified with alabeling group.
 23. The compound of claim 21 wherein R⁴ comprisesnitrogen.
 24. The compound of claim 21 wherein R⁴ is covalently linkedto a solid support.
 25. A method for modifying a nucleoside comprisingthe steps of: a) contacting a nucleoside having at least onehallogenomethyl-modified hydroxyl group with a thiosulfonate compoundthereby forming a thiosulfonated nucleoside; and b) contacting saidthiosulfonated nucleoside with a hydrocarbylthiol compound therebyforming a hydrocarbyldithiomethyl-modified nucleoside.
 26. The method ofclaim 25 wherein said thiosulfonate compound is selected from the groupconsisting of alkylthiosulfonate and arylthiosulfonate.
 27. The methodof claim 25 further comprising the step of labeling saidhydrocarbyldithiomethyl-modified nucleoside.
 28. A method for sequencinga nucleic acid comprising the steps of: a) contacting a target nucleicacid with a primer under conditions wherein said primer anneals to saidtarget nucleic acid in a sequence specific manner and wherein at least aportion of said primer is complementary to a portion of said targetnucleic acid; b) incorporating a hydrocarbyldithiomethyl-modifiednucleotide into said primer; and c) detecting incorporation of saidhydrocarbyldithiomethyl-modified nucleotide, wherein saidhydrocarbyldithiomethyl-modified nucleotide is complementary to saidtarget nucleic acid at said hydrocarbyldithiomethyl-modifiednucleotide's site of incorporation thereby identifying the sequence ofone nucleobase of said target nucleic acid.
 29. The method of claim 28wherein said incorporating step is catalyzed by a DNA polymerase. 30.The method of claim 28 wherein said sequencing method is selected fromthe group consisting of minisequencing and sequencing by synthesis. 31.The method of claim 28 wherein said method is effective for use with asequencing array.
 32. A method for sequencing a nucleic acid comprisingthe steps of: a) contacting a target nucleic acid with a primer underconditions wherein said primer anneals to said target nucleic acid in asequence specific manner and wherein at least a portion of said primeris complementary to a portion of said target nucleic acid; b)incorporating a first 3′-hydrocarbyldithiomethyl-modified nucleotideinto said primer; c) detecting said incorporation of said first3′-hydrocarbyldithiomethyl-modified nucleotide thereby identifying thesequence of a nucleobase of said target nucleic acid; d) removing saidhydrocarbyldithiomethyl group from said first incorporatedhydrocarbyldithiomethyl-modified nucleotide to form a first elongatedprimer having a free hydroxyl group; e) incorporating a second3′-hydrocarbyldithiomethyl-modified nucleotide into said first elongatedprimer; and f) detecting said second hydrocarbyldithiomethyl-modifiednucleotide thereby identifying the sequence of another nucleobase ofsaid target nucleic acid, wherein said first3′-hydrocarbyldithiomethyl-modified nucleotide and said second3′-hydrocarbyldithiomethyl-modified nucleotide are complementary to saidtarget nucleic acid at each said nucleotide's site of incorporation. 33.The method of claim 32 wherein said detecting steps are performed beforeremoving said hydrocarbyldithiomethyl group.
 34. The method of claim 32wherein said detecting steps are performed after removing saidhydrocarbyldithiomethyl group.
 35. The method of claim 32 wherein saidmethod is effective for use with a sequencing array.
 36. The method ofclaim 32 wherein steps a), b), c), d), e), and f) are performed underconditions that do not disrupt the annealing of said primer to saidtarget nucleic acid.
 37. A compound comprising the Formula:

wherein R¹ is a H, a protecting group, a phosphate, diphosphate, or atriphosphate, or a residue of a nucleic acid; R² is a nucleobase; R⁴, R⁵and R⁶ are together or separately H or hydrocarbyl; and R⁷ is H,H-phosphonate or phosphoramidite.
 38. An oligonucleotide synthesissupport comprising the formula:

wherein R¹ is H, phosphate, diphosphate, triphosphate, or a protectinggroup, R² is a nucleobase, R³ is H, OH, or a protected form of OH, and Zis a group effective for covalent attachment to a solid support, saidsolid support being effective for covalently bonding an oligonucleotideduring oligonucleotide synthesis.
 39. The support of claim 38 whereinsaid Z is selected from the group consisting of amino, amide, ester, andether.
 40. A method for synthesizing an oligonucleotide comprising thesteps of: a) providing a 5′ protected first nucleoside covalently bondedto a solid support through a linker; b) deprotecting said firstnucleoside at its 5′ position; c) covalently bonding another 5′protected nucleoside to said first nucleotide at the 5′ position of saidfirst nucleoside; d) deprotecting said another nucleoside at its 5′position; and e) repeating steps c) and d) for adding additionalprotected nucleosides, said linker securing said first nucleotide tosaid solid support via a hydrocarbyldithiomethyl bond.
 41. The method ofclaim 40 wherein said method is optimized for use in an array.
 42. Themethod of claim 40 wherein said method is effective for inverting saidoligonucleotide thereby forming an oligonucleotide having a free 3′hydroxyl and being covalently linked to a solid support.
 43. The methodof claim 42 wherein said method is optimized for use in an array. 44.The method of claim 40 further comprising the step of cleaving saidoligonucleotide from said solid support.
 45. A method for synthesizingan oligoribonucleotide comprising the steps of: a) providing a firstprotected ribonucleoside covalently bonded to a solid support; b)covalently linking at least one 2′-hydrocarbyldithiomethyl-modifiedribonucleoside to said first ribonucleoside to form anoligoribonucleotide; c) partially de-protecting said oligoribonucleotideunder acidic or basic conditions; and d) contacting saidoligoribonucleotide with a reducing agent under neutral conditionsthereby completely de-protecting said oligoribonucleotide, wherein saidmethod is effective for preventing cleavage or migration ofintemucleotide phosphate bonds, and wherein saidhydrocarbyldithiomethyl-modified ribonucleoside comprises ahydrocarbyldithiomethyl group bound at the 2′ position of saidhydrocarbyldithiomethyl-modified ribonucleoside.
 46. The method of claim45 wherein the pH of said neutral conditions ranges from about 5 toabout
 9. 47. The method of claim 46 wherein said pH is about
 7. 48. Themethod of claim 45 wherein said method is effective for inverting saidoligoribonucleotide thereby forming a solid phase bound oligonucleotidehaving a free 3′ hydroxyl.
 49. The method of claim 45 wherein said firstprotected ribonucleoside is secured to said solid support via ahydrocarbyldithiomethyl bond.
 50. A method for sequencing a nucleic acidcomprising the steps of: a) providing a primer array comprising aplurality of sequencing primers; b) contacting a target nucleic acidwith said primer array under conditions wherein said sequencing primersanneal to said target nucleic acid in a sequence specific manner therebyforming target-primer complexes between complementary portions of saidsequencing primers and said target nucleic acid; c) incorporating afirst 3′ -hydrocarbyldithiomethyl-modified nucleotide into at least onesequencing primer portion of said target-primer complexes, said first3′-hydrocarbyldithiomethyl-modified nucleotide being complementary tosaid target nucleic acid; and d) detecting said incorporation of saidfirst 3′-hydrocarbyldithiomethyl-modified nucleotide, wherein said first3′-hydrocarbyldithiomethyl-modified nucleotide is complementary to saidtarget sequence at said first 3′-hydrocarbyldithiomethyl-modifiednucleotide's site of incorporation.
 51. The method of claim 50 furthercomprising the steps of: e) removing said hydrocarbyldithiomethyl groupfrom said first incorporated 3′-hydrocarbyldithiomethyl-modifiednucleotide to form a first elongated target-primer complex having a free3′ hydroxyl group; f) incorporating a secondhydrocarbyldithiomethyl-modified nucleotide into said first elongatedtarget-primer complex; and g) detecting said second3′-hydrocarbyldithiomethyl-modified nucleotide, wherein said second3′-hydrocarbyldithiomethyl-modified nucleotide is complementary to saidtarget sequence at said second 3′-hydrocarbyldithiomethyl-modifiednucleotide's site of incorporation.
 52. The method of claim 50 whereinsaid detecting said incorporation step is performed before removing ahydrocarbyldithiomethyl moiety.
 53. The method of claim 50 wherein saidmethod is effective for producing a plurality of nucleotide sequences,said nucleotide sequences corresponding to overlapping nucleotidesequences of said target nucleic acid.
 54. The method of claim 51wherein said step e) is performed under conditions that do not disruptsaid target-primer complexes.
 55. A method for synthesizing anoligonucleotide comprising the steps of: a) providing a 5′ protectedfirst nucleoside covalently bonded to a solid support through ahydrocarbyldithiomethyl containing linker; b) deprotecting said firstnucleoside at its 5′ position; c) covalently bonding another 5′protected nucleoside to said first nucleotide at the 5′ position of saidfirst nucleoside; d) deprotecting said another nucleoside at its 5′position; e) optionally repeating steps c) and d) for adding additionalprotected nucleosides thereby producing an oligonucleotide; f)optionally selectively cleaving a protecting group from saidoligonucleotide thereby forming a partially deprotected oligonucleotide;g) selectively cleaving said hydrocarbyldithiomethyl containing linker;and h) isolating said partially deprotected oligonucleotide.
 56. Themethod of claim 55 further comprising the step of modifying the 3′terminus of said oligonucleotide with a reactive or detectable moiety.57. The method of claim 55 wherein at least one of said 5′ protectednucleosides comprises a hydrocarbyldithiomethyl moiety.